Fig 1: Loss of Psip1 leads to reduced Bmi1 and Ctbp1 on target genes. (A) Psip1/p75 complexes purified from stably transduced GFP-p75, and GFP (control) cells, and immunoblotted using antibodies against Mll1, Mll2, Bmi1, Ctbp1, Cbx4, Ezh2, Mel18, Rybp and Pcna. Note that 5% input extract were also loaded. (B) Mean Log2 ChIP/input for Bmi1, Cbx4, Ring1B and Ctbp1 in WT and Psip1-/- MEFs over HoxA (left) and HoxD clusters (right) using custom tiling arrays as in Figure 1. (C) Box plots showing Log2 ChIP/input distributions for Bmi1, Ctbp1, Cbx4 and Ring1B in WT and Psip1-/- MEFs over 5' and 3' regions of HoxA and HoxD clusters, and non-Hox genes (n = 2 biological replicates). Asterisk (*) indicates a significant difference in ChIP/input signal between WTand Psip1-/-(P < 0.01, Wilcoxon rank-sum test). (D) Sequential-ChIP qPCR over promoter (Hoxa9p), exon1 (Hoxa9E1) and exon 2 of Hoxa9 (Hoxa9E2) in Psip1 WT and Psip1-/- MEFs. First, ChIP was performed with covalently coupled IgG (IgG-Bmi1), p75 (p75-Bmi1) and Mll1 (Mll1-Bmi1), followed by Bmi1 antibodies for second ChIP. Schematic below shows the Hoxa9 gene and primers used for PCR. (E) ChIP qPCR for Bmi1 and p75 in WT (green) and Psip1-/- (orange) MEFS, and inPsip1-/- rescued with WT p75 (Psip1-/-p75 R; black) or p75 with W21A PWWP point mutation (Psip1-/-p75 W21A R; gray). Ptn promoter primers (Ptnp) were used as a control. Mean (+/- s.e.m.) percent (%) input bound (n = 3) are plotted.
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